The Mechanism Of ANLN In The Carcinogenesis Of Bladder Cancer

?Background? Bladder cancer is the most common malignant tumor of the urological system in China.Nearly fifty percent of patients are already at advanced stage at diagnosis,and the prognosis of these patients are poor.Bladder cancer is classified as non-muscle invasive bladder cancer(NMIBC)and muscle-invasive bladder cancer(MIBC)according to the pathological stage of bladder cancer.Patients with NMIBC is usually treated by transurethral resection of bladder tumor(TURBT),and patients with MIBC need undergo radical cystectomy.The prognosis of bladder cancer patients varies greatly,approximately 60% of patients with NMIBC undergo TURBT will relapse,and about 50% of patients with NMIBC undergo radical cystectomy will develop local recurrence or distant metastasis.Therefore,it is meaningful to explore the molecular mechanism that contributes to the carcinogenesis and progress of bladder cancer,and to explore the molecular classification of bladder cancer,which will help to improve the personalized treatment of bladder cancer.The latest high-through output sequencing technology and bioinformatics analysis have provided an efficient approach to investigate the molecular alteration that contributed to the carcinogenesis of bladder cancer,which will help to improve early diagnosis and personalized treatment.Currently,the transcriptome sequencing data of Chinese bladder cancer patients is still lacking.Our department has cooperation with the Beijing Genomics Institute since 2011,we aimed to investigate the carcinogenesis mechanism of bladder cancer via transcriptome sequencing and to explore biomarkers for predicting the prognosis of bladder cancer.?Objective? The study aimed to explore the molecular mechanism involved in the carcinogenesisand progress of bladder cancer via the second generation of transcriptome sequencing between ten pairs of bladder cancer samples and their corresponding adjacent normal tissue,and to explore biomarkers for molecular classification of bladder cancer and predicting the prognosis.?Materials and Methods? 1.Extract RNA from ten pairs of bladder cancer samples and their corresponding adjacent normal tissue for transcriptome sequencing.Select candidate genes that involved in the carcinogenesis of bladder cancer and could serve as potential biomarkers of prognosis vis bioinformatic analysis.2.Extract RNA from 40 pairs of bladder cancer samples and their corresponding adjacent normal tissue.The expression level of ANLN m RNA was validated within the 40 pair samples by q RT-PCR analysis.The relationship between the level of ANLN and pathological stage and grade of bladder cancer was analyzed.3.Data mining from the public database Gene Expression Omnibus(GEO)and The Cancer Genome Atlas(TCGA),to further explore the relationship between the level of ANLN and the pathological stage and prognosis.4.The expression of protein anillin which was encoded by ANLN in paraffin-embedded sections was evaluated by immunohistochemical staining.The expression level of anillin between bladder tumor and the matched normal epithelial tissue was investigated.The relationship between anillin expression and pathological parameters as well as the prognosis of patients were also explored.5.The expression level of ANLN was examined in seven strains of human bladder cancer cell lines.The ANLN expression was knock down by transient si RNA transfection in J82 and 5637 cells,which had the highest level of ANLN among 7 bladder cancer cell lines.The proliferation ability of J82 and 5637 cells after ANLN knock down was evaluated by CCK-8 kit.The ANLN stably knock downJ82 cells was developed by transfection of lenti-virus with sh RNA against ANLN.Subcutaneous nude mice model was constructed by injected J82 cells with lenti-sh ANLN or lenti-vector control subcutaneously into the lower flank of female nude mice at a concentration of 1 x 107 cells/ml.The intravesical orthotopic bladder cancer model was also developed to investigate the impact of ANLN on the proliferation of bladder cancer cells.6.Wound healing and transwell invasion was performed to evaluate the influence of ANLN on the migration and invasion ability of bladder cancer cells.7.Flow cytometry analysis was performed to investigate the impact of ANLN knockdown on the cell cycle and apoptosis.8.Immunofluorescence staining and hematoxylin-eosin staining cells was conducted to explore the impact of ANLN gene knockdown on mitosis and cell morphology in J82 cells.9.The differentially expressed genes in ANLN knockdown group and control group were analyzed by microarray analysis to explore the mechanism of ANLN involved in the carcinogenesis and progress of bladder cancer,and the interaction with other signaling pathways.?Results? 1.ANLN was identified as a candidate gene of interest for three reasons.Firstly,ANLN expression was up-regulated,with a log2-fold change of 2.926 and probability of 0.835,in tumor samples compared with corresponding normal tissues.The expression level,quantified by reads per gene per kilobase exon per million mapped reads,was up-regulated in 9 out of the 10 tumor samples.Secondly,ANLN was a potential biomarker for tumor staging due to its expression level increased with pathological stage of bladder cancer.Thirdly,after data-mining and literature search,ANLN was not previously reported to beassociated with bladder cancer.2.ANLN expression pattern was further validated by q RT-PCR in an additional 40 paired bladder cancer samples and matched normal epithelial tissue.ANLN expression was confirmed to be up-regulated in a majority of tumor samples(36/40),especially in MIBC,when compared with their corresponding normal tissues.3.We evaluated ANLN expression by immunohistochemical analysis in 209 patients who underwent cystectomy or TURBT.ANLN expression level was always lower in normal epithelial tissue than in tumor tissues.With a median follow-up of 27.9 months,patients with high ANLN expression in tumor showed significantly poorer cancer-specific(median,22.4 vs.37.3 months,p=0.001),progression-free(median,19.7 vs.27.9 months,p=0.001)and recurrence-free survival(median,17.1 vs.25.2 months,p=0.011)compared with patients with low expression.4.We analyzed GEO datasets GSE31684,which was published by Memorial Sloan-Kettering Cancer Center with 93 patients with high-risk BLCA,the ANLN expression level,on average,significantly increased as tumor stage progress.To validate these observations in our single institution,we analyzed the association between ANLN m RNA expression and clinical outcome in TCGA,patients with ANLN up-regulation had significantly poorer overall survival(median,14.75 months)compared with those who did not altered(median,36.86 months,p=0.007).5.CCK-8 cell proliferation assay result showed that the interference of ANLN expression in J82 and 5637 cells could inhibit the proliferation of bladder cancer cells compared with the negative control group.The subcutaneous nude mice bladder cancer model and the intravesical orthotopic bladder cancer model further confirmed that the ANLN gene knockdown reduced the proliferation rate ofbladder cancer cells.6.Would healing assay results showed that ANLN knockdown could inhibit the migration of J82 and 5637 cells compared with the negative control group.Transwell Matrigel invasion assay result suggested that ANLN knockdown could significantly inhibit the invasion ability of J82 and 5637 cells.7.Treatment with si-ANLN significantly increased cell percentage of G2 phase compared with control cells,which suggested that cells with ANLN knockdown were arrested at the G2 phase.In terms of apoptosis,there was no significant difference between two groups.Immunofluorescence analysis showed that less ANLN protein was observed to form the contract ring for nuclear division in ANLN knockdown J82 cells.J82 cells with ANLN knockdown and control cells were further stained by hematoxylin & eosin.Compared with control cells,multinucleated cancer cells were observed at higher frequency when ANLN was knockdown.8.We performed microarray analysis to compare the ANLN knockdown group and control group.In J82 cells,the majority of the top ten terms in GO biological process classification were closely related to regulation of locomotion and cell migration,which was consistent with the observation we showed in the above ANLN function analysis.Several tumorigenesis related pathways,e.g.Jak-STAT,Toll-like receptor,and TNF signaling pathways,PI3K/Akt signal pathway were found to be altered after ANLN knockdown,which suggested that the ANLN gene could have mutual interaction with these pathways,which warrant further investigation.?Conclusions? ANLN gene and its encoded protein were highly expressed in bladder cancer tissues,and the expression level of ANLN gene was closely correlated with thepathological grade and stage of bladder cancer.ANLN high expression is the independent high risk factor of bladder cancer prognosis.The interference of ANLN gene expression could significantly inhibit the proliferation,migration and invasion of bladder cancer cells.ANLN involved in the biological function of bladder cancer cells by regulating the mitosis and cell cycle of bladder cancer cells.The interaction ANLN and PI3K/AKT,TNF and other signaling pathways was closely related to tumorigenesis and progression of bladder cancer.